06-methylguanine (06MeGua) DNA adducts formed by N-nitrosoamines are mutagenic and carcinogenic. They are repaired by a stoichiometric transfer of the methyl group to an acceptor protein that inactivates the acceptor. Our recent data indicate that, in response to a single N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment that methylates the DNA and depletes the preexisting constitutive acceptor protein, MNNG-resistant (Mer+) cells rapidly resynthesize the lost activity whereas MNNG-sensitive (Mer-) cells are unable to restore this activity. Repetitive MNNG exposures of Mer+ cells result in the induction of acceptor activity to a level 2 to 3-fold above the constitutive level, and adaptive response absent in most Mer- cell lines tested. Hence, our data suggest that the Mer character of a cell line correlates mostly with its ability to respond to DNA alkylation with an induced synthesis of the repair activity. By using a simple in vitro assay, we have recently developed, that measures the amount of 06MeGua-acceptor protein in cell extracts, we shall study the regulation of the constitutive and induced 06MeGua repair activity and the nature of the Mer- phenotype in human cells in culture. We propose to determine: i) the nature of the trigger that leads to the synthesis of the induced activity; ii) the coinduction of other repair activities; iii) the effect of cell cycle on the constitutive level and the magnitude of the induced response; and iv) the effect of agents that influence gene expression. We also, propose v) to compare the biochemical properties of the constitutive and induced 06MeGua-acceptor proteins; vi) to study the role of RNA synthesis and the regulation of in vitro translation of mRNA; and vii to attempt to isolate Mer+ cell lines from Mer ones by a) mutagenizing; b) affecting gene expression; and c) transforming Mer- cells with DNA isolated from Mer+ cells. The proposed research will advance the understanding of how at a cellular level human cells regulate DNA repair after their DNA is damaged.